The Interplay Between DSL Proteins and Ubiquitin Ligases in Notch Signaling

Development (Cambridge, England). Sep, 2005  |  Pubmed ID: 16093323

Lateral inhibition is a pattern refining process that generates single neural precursors from a field of equipotent cells and is mediated via Notch signaling. Of the two Notch ligands Delta and Serrate, only the former was thought to participate in this process. We now show that macrochaete lateral inhibition involves both Delta and Serrate. In this context, Serrate interacts with Neuralized, a ubiquitin ligase that was heretofore thought to act only on Delta. Neuralized physically associates with Serrate and stimulates its endocytosis and signaling activity. We also characterize a mutation in mib1, a Drosophila homolog of mind bomb, another Delta-targeting ubiquitin ligase from zebrafish. Mib1 affects the signaling activity of Delta and Serrate in both lateral inhibition and wing dorsoventral boundary formation. Simultaneous absence of neuralized and mib1 completely abolishes Notch signaling in both aforementioned contexts, making it likely that ubiquitination is a prerequisite for Delta/Serrate signaling.

Role of Conserved Intracellular Motifs in Serrate Signalling, Cis-inhibition and Endocytosis

The EMBO Journal. Oct, 2006  |  Pubmed ID: 17006545

Notch is the receptor in a signalling pathway that operates in a diverse spectrum of developmental processes. Its ligands (e.g. Serrate) are transmembrane proteins whose signalling competence is regulated by the endocytosis-promoting E3 ubiquitin ligases, Mindbomb1 and Neuralized. The ligands also inhibit Notch present in the same cell (cis-inhibition). Here, we identify two conserved motifs in the intracellular domain of Serrate that are required for efficient endocytosis. The first, a dileucine motif, is dispensable for trans-activation and cis-inhibition despite the endocytic defect, demonstrating that signalling can be separated from bulk endocytosis. The second, a novel motif, is necessary for interactions with Mindbomb1/Neuralized and is strictly required for Serrate to trans-activate and internalise efficiently but not for it to inhibit Notch signalling. Cis-inhibition is compromised when an ER retention signal is added to Serrate, or when the levels of Neuralized are increased, and together these data indicate that cis-inhibitory interactions occur at the cell surface. The balance of ubiquitinated/unubiquitinated ligand will thus affect the signalling capacity of the cell at several levels.

In Vivo Imaging of Drosophila Melanogaster Pupae with Mesoscopic Fluorescence Tomography

Nature Methods. Jan, 2008  |  Pubmed ID: 18066071

We report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.

Exploiting Position Effects and the Gypsy Retrovirus Insulator to Engineer Precisely Expressed Transgenes

Nature Genetics. Apr, 2008  |  Pubmed ID: 18311141

A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.

Developmental Biology: Our Fly Cousins' Gut

Nature. Jul, 2008  |  Pubmed ID: 18668098

Homeostasis in Infected Epithelia: Stem Cells Take the Lead

Cell Host & Microbe. Oct, 2009  |  Pubmed ID: 19837370

To maintain tissue homeostasis and avoid disease, epithelial cells damaged by pathogens need to be readily replenished, and this is mainly achieved by the activation of stem cells. In this Short Review, we discuss recent developments in the exciting field of host epithelia-pathogen interaction in Drosophila as well as in mammals.

Synergy Between Bacterial Infection and Genetic Predisposition in Intestinal Dysplasia

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2009  |  Pubmed ID: 19934041

Accumulating evidence suggests that hyperproliferating intestinal stem cells (SCs) and progenitors drive cancer initiation, maintenance, and metastasis. In addition, chronic inflammation and infection have been increasingly recognized for their roles in cancer. Nevertheless, the mechanisms by which bacterial infections can initiate SC-mediated tumorigenesis remain elusive. Using a Drosophila model of gut pathogenesis, we show that intestinal infection with Pseudomonas aeruginosa, a human opportunistic bacterial pathogen, activates the c-Jun N-terminal kinase (JNK) pathway, a hallmark of the host stress response. This, in turn, causes apoptosis of enterocytes, the largest class of differentiated intestinal cells, and promotes a dramatic proliferation of SCs and progenitors that serves as a homeostatic compensatory mechanism to replenish the apoptotic enterocytes. However, we find that this homeostatic mechanism can lead to massive over-proliferation of intestinal cells when infection occurs in animals with a latent oncogenic form of the Ras1 oncogene. The affected intestines develop excess layers of cells with altered apicobasal polarity reminiscent of dysplasia, suggesting that infection can directly synergize with the genetic background in predisposed individuals to initiate SC-mediated tumorigenesis. Our results provide a framework for the study of intestinal bacterial infections and their effects on undifferentiated and mature enteric epithelial cells in the initial stages of intestinal cancer. Assessment of progenitor cell responses to pathogenic intestinal bacteria could provide a measure of predisposition for apoptotic enterocyte-assisted intestinal dysplasias in humans.

Embryonic Multipotent Progenitors Remodel the Drosophila Airways During Metamorphosis

Development (Cambridge, England). Nov, 2010  |  Pubmed ID: 20940225

Adult structures in holometabolous insects such as Drosophila are generated by groups of imaginal cells dedicated to the formation of different organs. Imaginal cells are specified in the embryo and remain quiescent until the larval stages, when they proliferate and differentiate to form organs. The Drosophila tracheal system is extensively remodeled during metamorphosis by a small number of airway progenitors. Among these, the spiracular branch tracheoblasts are responsible for the generation of the pupal and adult abdominal airways. To understand the coordination of proliferation and differentiation during organogenesis of tubular organs, we analyzed the remodeling of Drosophila airways during metamorphosis. We show that the embryonic spiracular branch tracheoblasts are multipotent cells that express the homeobox transcription factor Cut, which is necessary for their survival and normal development. They give rise to three distinct cell populations at the end of larval development, which generate the adult tracheal tubes, the spiracle and the epidermis surrounding the spiracle. Our study establishes the series of events that lead to the formation of an adult tubular structure in Drosophila.